The invention relates generally to polypeptides and complexes of two or more polypeptides, as well as to methods of use thereof.
Most, if not all, biologically important activities are mediated at the tissue, cellular, and subcellular level at least in part by interactions between one or more proteins. These biologically important activities can include, e.g., cell division, cell differentiation, anabolic activities, and catabolic activities. Interacting proteins or polypeptides can form a complex. Accordingly, failure to form a given polypeptide complex can result in deleterious consequences to a cell or individual. Conversely, the inappropriate formation of a given polypeptide complex can likewise be undesirable.
The identification of protein complexes associated with specific biological activities can be used to identify or prevent conditions associated with the absence or presence of these complexes.
The invention is based, in part, upon the identification of protein-protein interactions in the yeast S. cerevisiae and humans. Interacting proteins present in complexes according to the invention are shown in, e.g., Table 3.
In one aspect, the invention provides a purified complex including a first polypeptide that includes the amino acid sequence encoded by the open reading frame (xe2x80x9cORFxe2x80x9d) listed in Table 3, column 1, and a second polypeptide that includes the amino acid sequence of the corresponding polypeptide encoded by the ORF recited in column 5 of Table 3. Gene names for the ORFs recited in Table 3, column 1, and Table 3, column 5 are provided in Table 3, columns 2 and 6, respectively.
In another aspect, the invention provides a purified complex including a first polypeptide and a second polypeptide selected from, or including, the human polypeptides recited in Table 7, column 2, and the corresponding polypeptides recited in Table 7, column 6. Complexes of polypeptides including the binding domains of such polypeptides, and complexes having labeled polypeptide, are also provided.
The invention also provides purified complexes of a first and a second polypeptide. The first polypeptide is a polypeptide functionally classified in the MIPS database as a Cell/Growth/Cell Division/DNA Synthesis protein; a Cell Rescue/Cell Defense/Cell Death and Aging Protein; a Cellular Biogenesis protein; a Cellular Organization protein; a Classification Not-Yet Clear Cut protein; an Energy Protein; an Intracellular Transport protein; an Ionic Homeostasis protein, a Metabolism protein; a Protein Destination protein; a Protein Synthesis protein; a Retrotransposon/Plasmid protein; a Signal Transduction protein; a Transcription protein; a Transport Facilitation protein, or an Unclassified protein. The second polypeptide is the corresponding polypeptide recited in Table 3, column 5 or Table 7, column 6, respectively.
The invention also provides a purified complex of a first and second polypeptide, where at least one of the polypeptides is a microtubule or microtubule-associated protein, a heme biosynthesis protein, or a cell wall or cell-wall synthesis protein.
The invention further provides purified chimeric complexes including a yeast polypeptide and a human ortholog polypeptide. In some embodiments the yeast polypeptide includes the amino acid sequence of the polypeptides recited in Table 7, column 1, and the human polypeptide includes the amino acid sequence of the corresponding polypeptides recited in Table 7, column 6. In other embodiments the yeast polypeptide is selected from, or includes, the polypeptides recited in Table 7, column 5, and the human ortholog polypeptide is selected from, or includes, the polypeptides recited in Table 7, column 2.
In a further aspect, the invention provides chimeric polypeptides having six or more amino acids of a first polypeptide covalently linked to six or more amino acids of a second polypeptide. In some embodiments, the chimeric polypeptides are yeast-yeast chimeras, while in others the chimeric polypeptides are human-human or yeast-human chimera. In some embodiments, the first polypeptide is selected from the polypeptides recited in Table 3, column 1, and the second polypeptide is selected from the polypeptides recited in Table 3, column 5. In other embodiments, the first polypeptide is selected from polypeptides recited in Table 7, columns 1 or 2, and the second polypeptide is selected from the polypeptides recited in Table 7, columns 5 or 6. Nucleic acids encoding chimeric polypeptides, and vectors and cells containing the same, are also provided.
In yet another aspect, the invention provides an antibody which specifically binds polypeptide complexes according to the invention. The antibody preferably binds to a complex comprising one or more polypeptides with greater affinity than its affinity for either polypeptide that is not present in the complex.
Also provided by the invention are kits containing reagent which can specifically detect the complexes of the invention. In one embodiment, the reagent is a complex-specific antibody, while in other embodiments the reagent is an antibody specific for the first or second polypeptides of the complex.
In another aspect, the invention provides pharmaceutical compositions including the complexes described herein. Such compositions are formulated to be suitable for therapeutic administration in the treatment of deficiencies or diseases involving altered levels of the complexes of the invention.
In still another aspect, the invention provides methods of identifying an agent which disrupts a polypeptide complex by providing a complex described herein, contacting the complex with a test agent, and detecting the presence of a polypeptide displaced from the complex. In certain embodiments, the complex includes at least one polypeptide comprising a microtubule or microtubule-associated protein, a heme biosynthesis protein, or a cell wall or cell-wall synthesis protein.
In a further aspect, the invention provides a method for inhibiting the interaction of a protein with a ligand by contacting a complex of the protein and ligand with an agent that disrupts the complex. In certain embodiments, the protein is a microtubule or microtubule associated protein, a heme biosynthesis protein, or a cell wall or cell-wall synthesis protein, and the ligand is a corresponding interacting polypeptide described herein.
In yet another aspect, the invention provides a method of identifying a polypeptide complex in a subject by providing a biological sample from the subject and detecting, if present, the level of a complex, described herein, in the subject.
Also provided by the invention is a method for detecting a polypeptide in a biological sample by providing a biological sample containing a first polypeptide, and contacting the sample with a second polypeptide under conditions suitable to form a polypeptide complex.
In another aspect, the invention provides a method for removing a first polypeptide from a biological sample by providing a biological sample including the first polypeptide, contacting the sample with a second polypeptide under conditions suitable for formation of a polypeptide complex, and removing the complex, thereby effectively removing the first polypeptide. In certain embodiments, the first polypeptide is selected from, or includes, the polypeptides recited in Table 7, column 2, and the second polypeptide is selected from, or includes, the polypeptides recited in Table 7, column 6. In another embodiment, the first polypeptide is selected from, or includes, the polypeptides recited in Table 7, column 6, and the second polypeptide is selected from, or includes, the polypeptides recited in Table 7, column 2.
In a further aspect, the invention provides a method for determining altered expression of a polypeptide in a subject by providing a biological sample from the subject, measuring the level of polypeptide complex in the sample, and comparing the level of the complex in the sample to the level of complex in a reference sample with a known polypeptide expression level.
In a still further aspect, the invention provides a method of treating or preventing a disease or disorder involving altered levels of a complex described herein or a polypeptide described herein, by administering, to a subject in need thereof, a therapeutically-effective amount of at least one molecule that modulates the function of the complex or polypeptide. In one embodiment, the agent modulates the function of a polypeptide selected from the polypeptides recited in Table 7, columns 2 or 6.
In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications cited in this specification are incorporated by reference herein in their entirety.
The invention provides complexes of interacting polypeptides which have not heretofore been shown to interact directly, as well as methods of using these complexes.
Some interacting polypeptides were identified by identifying which of the predicted open-reading frames (ORFs) of the yeast S. cerevisiae encode polypeptides that interact in a yeast two-hybrid system. In one screen, 692 discrete interacting protein pairs were discovered. These interacting pairs include (i) interactions that place functionally unclassified proteins in a biological context, (ii) novel interactions between proteins involved in the same biological function, and (iii) novel interactions that link together biological functions into larger cellular processes.
A summary of the screening used to identify interacting yeast ORFS is shown in Table 1.
Table 2 indicates that the interacting proteins disclosed herein can be grouped by functional roles using the Munich Information Center for Protein Sequences (xe2x80x9cMIPSxe2x80x9d) classification system.
Some newly disclosed interactions place functionally unclassified proteins from the yeast genome in a biological context. For example, two proteins of unknown function, YGR010Wp and YLR328Wp (77% identical), were observed to interact with each other, and also to bind to omithine aminotransferase (Car2p), which catalyzes a step in arginine metabolism. This observation suggests that YGR010WP and YLR328Wp are implicated in arginine metabolism. In addition, because YGR010Wp and YLR328Wp are 40% identical to the human protein KIAA0479 (Genbank accession number AB007948), the interactive data further suggest that the human protein KIAA0479 is also involved in arginine metabolism.
Also included in the interactions are complexes of two or more proteins involved in functional pathways for which direct interactions have not been described previously. For example, proteins involved in autophagy, e.g., Apg13p, are shown herein to interact with proteins of the Cvt (cytoplasm-to-vacuole targeting) pathway, e.g., Lap4p. Previously, direct interactions between proteins involved in autophagy and the Cvt pathway had not been reported. Autophagy is a degradation pathway used under conditions of nutrient stress to non-selectively recycle cytoplasmic proteins and organelles to their constituent components, while the Cvt pathway is a biosynthetic pathway that transports the vacuolar enzyme aminopeptidase I (API, encoded by LAP4) specifically to the vacuole. See Scott et al., Curr. Opin. Cell. Biol. 10: 523 (1998). Several mutants in the Cvt pathway (cvt) and autophagocytosis (aut and apg) are allelic, suggesting that both pathways utilize some of the same molecular components. See Tsukada et al., FEBS Letters 333: 169 (1993); Thumm et al., FEBS Letters 349: 275 (1994); Harding et al., J. Cell. Biol. 131: 17621 (1996); Scott et al., Proc. Natl. Acad. Sci. USA 93: 12304 (1996).
A number of ORFs encoding proteins of unknown functions have been identified as components of autophagy. Since several of the genes altered in apg, aut, and cvt mutants have not yet been cloned, ORFs found in these interactions can be examined to determine if they encode any of these altered genes. It has also been shown that Lap4p is a self-interactor, corroborating previous evidence that Lap4p assembles into a dodecamer (see Funakoshi et al., Gene 192: 207 (1997)), and the observed interaction between Apg1 and Apg13 lends support to previous genetic evidence suggesting that APG1 is a high-copy suppressor of apg13 (Kim et al, J. Cell. Biol. 137: 609 (1997)).
An interaction was also identified between YDR201 Wp and YKR037Cp, two proteins known to be localized to the spindle pole body by mass spectrometry. See Wigge et al., J. Cell Biol. 141: 967 (1998). The interaction of these proteins may indicate their involvement in the regulation of mitotic events.
New insights into novel interactions between proteins involved in the same biological function are also provided. For example, the nuclear polyadenylated RNA-binding proteins Nab2p and Nab4p bind to the 3xe2x80x2 end of mRNA, but have distinct roles. See Kessler et al., Genes Dev. 11: 2545 (1997). Nab2p is required for the regulation of poly(A) tail length and export of mRNA from the nucleus, and Nab4p is essential for the cleavage of pre-mRNA at the correct 3xe2x80x2 site. The newly described interaction between Nab2p and Nab4p suggests that they may act in concert.
Similarly, in yeast, diverse cyclins bind to Cdc28p in a coordinated manner to modulate its kinase activity during the cell cycle. The B-type cyclins play a critical role in the induction of bipolar mitotic spindle formation. See Nasmyth, Curr. Opin. Cell. Biol. 5: 166 (1993). Each of the B-type cyclins, Clb1p, Clb2p and Clb3p, has presently been observed to form a complex with Cks1p and Cdc28p. The identification of interactions between Cks1p and each of Clb1p, Clb2p and Clb3p, suggests that the kinase activity of Cdc28p could be mediated by cyclin Bs through their interaction with Cks1p.
In another example, Ypt53p, a rab5-like GTPase involved in vacuolar protein sorting and endocytosis, has presently been shown to interact with Siw4p, a putative tyrosine phosphatase which acts in a complex to control nutrient-dependent cell proliferation. See Singer-Kruger et al., J. Cell. Biol. 125: 283 (1994); Saul et al., Gen. Microbiol. 131: 1797 (1985). One possible explanation for the observed interaction is that Ypt53p senses nutrient availability to coordinate cell cycle progression.
The newly identified protein-protein interactions connect biological functions into larger cellular processes. For example, the nuclear pore complex (NPC), consisting of as many as 50 different subunits, is the macromolecular-conducting channel between the nucleus and the cytoplasm. See Fabre et al., Ann. Rev. Genet. 31: 277 (1997); Marelli et al., J. Cell Biol. 143: 1813 (1998). Two newly identified NPC components, Nup53p and Nup59p/Asm4p, interact with Ndc1p, a protein required for spindle pole body (SPB) duplication and component of the nuclear envelope. Evidence of a physical interaction between components of the NPC and SPB suggests that these two structures located in the nuclear envelope may coordinate communication between the nucleus and the cytoplasm.
Another interaction involves the meiosis-specific protein, Msh5p, which is required for the resolution of cross-overs during meiosis. Hollingsworth et al., Genes Dev. 9: 1728 (1995). Meiotic recombination is initiated by double-strand breaks (DSBs), a prerequisite to cross-over formation that is resolved in a structure called the synaptonemal complex (SC). Mre11p is part of a complex that participates in DSB formation. See Usui et al., Cell 95: 705 (1998). It is also known that Tid3p helps form the spindle pole body and interacts with Dmc1p, a protein required for the formation of the SC. See Bishop et al., Cell 69: 439-56 (1992). It has presently been shown that Msh5p interacts with both Mre11p and Tid3p. These novel associations tie DSB formation and the resolution of cross-overs with Msh5p as the linking protein.
Similarly, to exit the cell cycle, cells must undergo a series of checkpoints that monitor correct microtubule and spindle formation. See Guenette et al., J. Cell. Sci. 108: 195 (1995). The present invention identifies at least two interactions that tie cycle regulation to microtubule assembly. The first is between a microtubule checkpoint protein, Bub3p and a spindle pole body checkpoint protein, Mad3p. This observation mirrors the recent interaction described between the human homologs of Bub3p and Mad3p. See Hoyt et al., Cell 66: 507-17 (1991); Hwang et al., Science 279. 1041 (1998); Taylor et al., J. Cell Biol. 142: 1 (1998). Interestingly, the second is between Mad3p and a known regulator of the Cdc28p kinase, Cln3p, See Cvrckova et al., EMBO J. 12: 5277 (1993). These interactions could give rise to a cascade Bub3xe2x86x92Mad3xe2x86x92Cln3pxe2x86x92Cdc28p, and may suggest a pathway to propagate the signal of incorrect microtubule formation during early events at the cell cycle arrest in G1 phase.
The complexes disclosed herein are useful, inter alia, in identifying agents which modulate cellular processes in which one or more members of the complex have previously been associated. For example, interacting Pro-Pairs 1a-1b (representing open reading frames YGR108W and YBR135W, or genes CLB1 and CKS1, respectively) as shown in Table 3, have both been previously implicated in cell growth, cell division, and/or DNA synthesis. Accordingly, new agents which modulate cell growth, cell division, and/or DNA synthesis can be identified by evaluating the ability of a test agent to affect formation or dissolution of a complex of the Pro-Pairs 1a and 1b.
Complexes according to the invention can also be used in methods for identifying a desired polypeptides in a biological sample by forming a complex of a first polypeptide and a second polypeptide that interacts with the first polypeptide. The presence of the complex indicates that the sample contains the first polypeptide.
These utilities, as well as additional utilities, are discussed in greater detail below
In one aspect, the invention includes a purified complex that includes two or more polypeptides. In one embodiment, the invention provides purified complexes of two or more polypeptides. One of the polypeptides includes a polypeptide selected from the polypeptides recited in Table 3, column 1 (referenced as ProPair 1a-692a) and another includes a polypeptide selected from the polypeptides recited in Table 3, column 5 (referenced as ProPair 1b-692b). In some embodiments the first and second polypeptides of the complex are the polypeptides enumerated in Table 3. In some embodiments a first polypeptide is listed as a xe2x80x9cbaitxe2x80x9d polypeptide and a second polypeptide is denoted as xe2x80x9cpreyxe2x80x9d polypeptide while in other embodiments the first polypeptide corresponds to a xe2x80x9cpreyxe2x80x9d polypeptide and the second is a xe2x80x9cbaitxe2x80x9d polypeptide.
By xe2x80x9ccorresponding polypeptidexe2x80x9d is meant, with reference to Tables 3-7, the polypeptide recited in the same row, reading across from left-to-right or right-to-left, as a specific selected peptide. For example, in Table 3, in the first row, the corresponding polypeptide of YGR108W is YBR135W. These protein pairs are designated as 1a and 1b, as is indicated in Table 3.
Similarly, in the first row, the corresponding polypeptide of YBR135W (ProPair 1b) is YBR108W. (ProPair 1a). In the second row, however, the corresponding polypeptide of YBR135W (a prey protein; ProPair 2b) is YPR119W (a bait protein; ProPair 2a).
Also as used herein, xe2x80x9cproteinxe2x80x9d and xe2x80x9cprotein complexxe2x80x9d are used synonymously with xe2x80x9cpolypeptidexe2x80x9d and xe2x80x9cpolypeptide complex.xe2x80x9d A xe2x80x9cpurifiedxe2x80x9d polypeptide, protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the polypeptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language xe2x80x9csubstantially free of cellular materialxe2x80x9d includes preparations of protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language xe2x80x9csubstantially free of cellular materialxe2x80x9d includes preparations of polypeptide complex having less than about 30% (by dry weight) of non-complex proteins (also referred to herein as a xe2x80x9ccontaminating proteinxe2x80x9d), more preferably less than about 20% of contaminating protein, still more preferably less than about 10% of contaminating protein, and most preferably less than about 5% non-complex protein. When the polypeptide or complex is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
In certain embodiments, the first polypeptide is labeled. In other embodiments, the second polypeptide is labeled, while in some embodiments, both the first and second polypeptides are labeled. Labeling can be performed using any art recognized method for labeling polypeptides. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, xcex2-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.
The invention also includes complexes of two or more polypeptides in which at least one of the polypeptides is present as a fragment of a complex-forming polypeptide according to the invention. For example, one or more polypeptides may include an amino acid sequence sufficient to bind to its corresponding polypeptide. A binding domain of a given first polypeptide can be any number of amino acids sufficient to specifically bind to, and complex with, the corresponding second polypeptide under conditions suitable for complex formation. The binding domain can be the minimal number of amino acids required to retain binding affinity, or may be a larger fragment or derivative of the polypeptides listed in Table 3, columns 1 and 4. Procedures for identifying binding domains can be readily identified by one of ordinary skill in the art and the procedures described herein. For example, nucleic acid sequences containing various portions of a xe2x80x9cbaitxe2x80x9d protein can be tested in a yeast two hybrid screening assay in combination with a nucleic acid encoding the corresponding xe2x80x9cpreyxe2x80x9d protein.
In certain embodiments, the xe2x80x9cbaitxe2x80x9d polypeptides of the complex are polypeptides categorized, for example, as a xe2x80x9cMetabolismxe2x80x9d protein in the MIPS database. In some embodiments, the xe2x80x9cpreyxe2x80x9d protein of the complex is also a xe2x80x9cMetabolismxe2x80x9d protein, while in other embodiments the xe2x80x9cpreyxe2x80x9d protein is, for example, an xe2x80x9cUnclassifiedxe2x80x9d protein (see Table 3; e.g., ProPair 195a-310a and ProPair 195b-310b). Other MIPS categories include, e.g., xe2x80x9cCell Growth/Cell Division/DNA Synthesisxe2x80x9d proteins (see Table 2).
In other embodiments, the complexes are human ortholog complexes, chimeric complexes, or specific complexes implicated in fungal pathways, as discussed in detail below.
Polypeptides forming the complexes according to the invention can be made using techniques known in the art. For example, one or more of the polypeptides in the complex can be chemically synthesized using art-recognized methods for polypeptide synthesis. These methods are common in the art, including synthesis using a peptide synthesizer. See, e.g., Peptide Chemistry, A Practical Textbook, Bodasnsky, Ed. Springer-Verlag, 1988; Merrifield, Science 232: 241-247 (1986); Barany, et al, Intl. J. Peptide Protein Res. 30: 705-739 (1987); Kent, Ann. Rev. Biochem. 57:957-989 (1988), and Kaiser, et al, Science 243: 187-198 (1989).
Alternatively, polypeptides can be made by expressing one or both polypeptides from a nucleic acid and allowing the complex to form from the expressed polypeptides. Any known nucleic acids that express the polypeptides, whether yeast or human (or chimerics of these polypeptides) can be used, as can vectors and cells expressing these polypeptides. Sequences of yeast ORFs and human polypeptides as referenced in Tables 3 and 7 are publicly available, e.g. at the Saccharomyces Genome Database (SGD) and GenBank (see, e.g. Hudson et al., Genome Res. 7: 1169-1173 (1997). If desired, the complexes can then be recovered and isolated.
Recombinant cells expressing the polypeptide, or a fragment or derivative thereof, may be obtained using methods known in the art, and individual gene product or complex may be isolated and analyzed (See, e.g., e.g., as described in Sambrook et al., eds., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausubel, et al., eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons, New York, N.Y., 1993). This is achieved by assays that are based upon the physical and/or functional properties of the protein or complex. The assays can include, e.g., radioactive labeling of one or more of the polypeptide complex components, followed by analysis by gel electrophoresis, immunoassay, cross-linking to marker-labeled products. Polypeptide complex may be isolated and purified by standard methods known in the art (either from natural sources or recombinant host cells expressing the proteins/protein complex). These methods can include, e.g., column chromatography (e.g., ion exchange, affinity, gel exclusion, reverse-phase, high pressure, fast protein liquid, etc), differential centrifugation, differential solubility, or similar methods used for the purification of proteins.
The invention further provides complexes of polypeptides useful, inter alia, in identifying agents that inhibit the growth of microorganisms such as fungi.
Human fungal infections have increased dramatically in incidence and severity in recent years. Advances in surgery and cancer treatments as well as the increasing use of broad-spectrum antimicrobials and the spread of HIV have increased the number of patients at risk for fungal infections. Most fungi are completely resistant to conventional antibacterial drugs.
The antifungal drugs presently available fall into several categories depending on their mode of action, as discussed below. Because several complexes according to the invention include proteins associated with these modes of action, the complexes can be used to identify anti-fungal agents.
Protein interactions which are useful for identifying anti-fungal agents are considered below.
(i) Interference in Nuclear Division
Griseofulvin interferes with nuclear division in fungal mitosis by disrupting the mitotic spindle and inhibiting cytoplasmic microtubule aggregation by interacting with polymerized microtubules. There is evidence that griseofulvin binds to a microtubule-associated protein in addition to binding to tubulin.
In accordance with the present invention, several interactions have presently been identified where one of the interacting partners is a microtubule or a microtubule-associated protein. Inhibiting any of these interactions could lead to the disruption of microtubules and interference in mitotic division, similar to the mode of action of griseofulvin, thereby providing a new means of inhibiting fungal activity. Accordingly, in some embodiments, the invention provides purified complexes of the proteins detailed in Table 4, below (interacting protein pairs are in bold, by row; a description of each protein follows).
As described above, in certain embodiments of these complexes contain the binding domains, of the polypeptides recited in Table 4, while other embodiments contain conservative variants of these polypeptides, or polypeptides which contain the polypeptides recited in Table 4.
(ii) Disruption of Ergosterol Biosynthesis
Azoles are synthetic compounds that can be classified as imidazoles (ketoconazole, clotrimazole and miconazole) or triazoles (itraconizole and fluconazole). The antifungal activity of azole drugs result from their reduction in the biosynthesis of ergosterol, the main sterol in the cell membranes of fungi. Reduction of ergosterol alters the structure of the cytoplasmic membrane as well as the function of several membrane-bound enzymes (such as those involved in nutrient transport and chitin synthesis). The azole drugs reduce ergosterol synthesis by inhibiting the fungal cytochrome p450 enzymes, specifically they inhibit the sterol 14-alpha-demethylase, a microsomal cytochrome P450-dependent enzyme system, leading to a decrease in ergosterol and an accumulation of 14-alpha-methylsterols. There is some evidence that the primary target of the azoles is the heme protein, which cocatalyzes cytochrome P-450-dependent 14-alpha-dependent 14-alpha-demethylation of lanosterol. One interaction containing a heme biosynthesis protein has been presently been identified (Table 5). Disruption of this interaction could also lead to depletion of ergosterol and accumulation of sterol precursors, including 14-alpha-methylated sterols, forming a membrane with altered structure and function. Accordingly, in some embodiments, the invention provides a purified complex of the proteins recited in Table 5, below.
Complexes containing one or more variants of these polypeptides are within the scope of the present invention, as are polypeptides having amino acid sequences which include the polypeptides recited in Table 5.
(iii) Cell Wall Synthesis Inhibition
Fungi share many biochemical targets with other eukaryotic cells. However, the fungal cell wall is a unique organelle and contains compounds, such as mannan, chitin and glucans, that are unique to fungi. The cell wall is dynamic and essential to the viability of the fungi due to its roles in osmotic protection, transport of macromolecules, growth, conjugation and spore formation. Major disruption of the composition or organization of the cell wall deleteriously affects cell growth. A number of compounds have been discovered that inhibit the development of fungal cell walls. Two class of these antifungal drugs are echinocandins, which inhibit glucan synthesis, and nikkomycins, which inhibit chitin synthesis.
Several interactions between proteins localized to the cell wall or enzymes responsible for production of cell wall components have presently been identified. Inhibiting any of these interactions could lead to a disruption of the cell wall, hence providing new means for inhibiting fungal viability. Accordingly, in certain embodiments, the present invention provides purified complexes of the proteins detailed in Table 6, below.
Embodiments of these complexes containing the binding domains or conservative variants of these polypeptides are within the scope of the present invention, as are polypeptides which contain the polypeptides recited in Table 6.
The invention also provides purified complexes of two or more human polypeptides. In some embodiments, the interacting polypeptides are human orthologs of the interacting yeast polypeptides. Human orthologs according to the invention are set out in Table 7, below.
Complexes of human ortholog binding domains, conservative variants, and polypeptides including the human orthologs recited in Table 7, are within the scope of the invention, as are labeled ortholog complexes and/or polypeptides.
The human polypeptides disclosed in Table 7 are related as orthologs to yeast polypeptides that interact to form complexes according to the invention. Table 7 reflects this relationship and specifies a yeast accession number for a given human ortholog. In particular. Table 7 includes in column 1 the yeast accession number for the yeast xe2x80x9cbaitxe2x80x9d sequence corresponding to the indicated human ortholog. Columns 2-4 provide the accession number of the human ortholog, the name of the human ortholog, and a description of the human ortholog, respectively, of the yeast xe2x80x9cbait sequencexe2x80x9d. Column 5 of Table 7 provides the yeast accession number of the yeast xe2x80x9cpreyxe2x80x9d sequence. Columns 6-8 provide the accession number of the human ortholog, the name of the human ortholog, and a description of the human ortholog, respectively, of the yeast xe2x80x9cprey sequencexe2x80x9d.
The Yeast and Human Ortholog Accession Numbers (ACCNO) listed in Table 7 are shown with their corresponding Sequence Identification Numbers (SEQIDNO) in Table 8.
In certain embodiments, one of the ortholog polypeptides includes a xe2x80x9cbaitxe2x80x9d polypeptide selected from the polypeptides recited in Table 7, column 2, and the other ortholog polypeptide includes a xe2x80x9cpreyxe2x80x9d protein selected from the polypeptides recited in Table 7, column 6. The yeast orthologs of these proteins are set out in columns 1 and 4 of Table 7, respectively. In some embodiments the first and second polypeptides of the complex are the polypeptides enumerated in Table 7. In some embodiments a first polypeptide is a xe2x80x9cbaitxe2x80x9d polypeptide and a second polypeptide is xe2x80x9ctargetxe2x80x9d polypeptide, while in other embodiments the first polypeptide is a xe2x80x9ctargetxe2x80x9d polypeptide and the second is a xe2x80x9cbaitxe2x80x9d polypeptide. Conservative variants of either polypeptide which retain binding specificity are within the scope of the invention, as are labeled forms of the complexes, as described above.
In other embodiments, the polypeptides are the binding domains of the xe2x80x9cbaitxe2x80x9d and xe2x80x9cpreyxe2x80x9d polypeptides listed in Table 7. A binding domain of a given first polypeptide may be any number of amino acids sufficient to specifically bind to, and complex with, the corresponding second polypeptide under conditions suitable for complex formation. A binding domain may be the minimal number of amino acids required to retain binding affinity, or may be a larger fragment or derivative of the polypeptides listed in Table 7, columns 2 and 6.
In certain embodiments, the xe2x80x9cbaitxe2x80x9d polypeptides of the ortholog complex are polypeptides categorized, for example, as a xe2x80x9cMetabolismxe2x80x9d protein in the MIPS database. In some embodiments, the xe2x80x9cpreyxe2x80x9d protein of the complex is also a xe2x80x9cMetabolismxe2x80x9d protein, while in other embodiments the xe2x80x9cpreyxe2x80x9d protein is, for example, an xe2x80x9cUnclassifiedxe2x80x9d protein (see Table 7). Other exemplary MIPS categories include, e.g., xe2x80x9cCell Growth/Cell Division/DNA Synthesisxe2x80x9d proteins (see Table 2).
In a further aspect, the invention provides chimeric polypeptide complex that includes at least one yeast polypeptide and at least one human ortholog of the corresponding interacting yeast polypeptide. In one embodiment, there is provided a purified chimeric complex including a yeast xe2x80x9cbaitxe2x80x9d polypeptide selected from the polypeptides recited in Table 7, column 1 and a human ortholog of the corresponding yeast xe2x80x9cpreyxe2x80x9d polypeptide; the human ortholog is selected from the polypeptides recited in Table 7, column 6 (while the corresponding yeast xe2x80x9cpreyxe2x80x9d proteins are recited in column 5). For example, with reference to Table 7, first row, in one embodiment, a chimeric protein containing YAL032C and P16118 is provided (P16118 is the human ortholog of corresponding yeast xe2x80x9cpreyxe2x80x9d protein YLR345W).
In other embodiments, the complex contains a human ortholog of a yeast xe2x80x9cbaitxe2x80x9d protein and a yeast xe2x80x9cpreyxe2x80x9d protein. The yeast xe2x80x9cpreyxe2x80x9d protein is selected from the polypeptides recited in Table 7, column 5, and the human ortholog of the corresponding yeast xe2x80x9cbaitxe2x80x9d protein is selected from the polypeptides recited in Table 7, column 2 (while the corresponding yeast xe2x80x9cbaitxe2x80x9d proteins themselves are recited in column 1). For example, with reference to Table 7, first row, in one embodiment, a chimeric protein containing Q13573 and YLR345W is provided (Q13573 is the human ortholog of corresponding yeast xe2x80x9cbaitxe2x80x9d protein YAL032C).
In certain embodiments the first and second polypeptides of the chimeric complex are the polypeptides recited in Table 7, columns 1 and 6, or columns 2 and 5, respectively, while in other embodiments, the polypeptides of the chimeric complex contain the polypeptides recited in Table 7. Conservative variants of either polypeptide which retain binding specificity are within the scope of the invention, as are labeled forms of the chimeric complexes, and chimeric complexes of binding domains, as described above.
In a further aspect, the invention provides a chimeric polypeptide that includes sequences of two interacting proteins according to the invention. The interacting proteins can be, e.g., the interacting protein pairs disclosed in Tables 3-7, herein. Also included are chimeric polypeptides including multimers, i.e., sequences from two or more pairs of interacting proteins. An example of such a chimeric polypeptide is a polypeptide that includes amino acid sequences from ProPair 1a and 1b, and from ProPair 2a and 2b. The chimeric polypeptide includes a region of a first protein covalently linked, e.g. via peptide bond, to a region of a second protein. In certain embodiments, the second protein is a species ortholog of the first protein. In some embodiments, the chimeric polypeptide contains regions of first and second proteins from yeast, where the proteins are selected from the xe2x80x9cbaitxe2x80x9d and corresponding xe2x80x9cpreyxe2x80x9d proteins recited in Table 3, columns 1 and 4, respectively. In other embodiments, the chimeric polypeptide contains regions of first and second human ortholog proteins, where the proteins are selected from the xe2x80x9cbaitxe2x80x9d and corresponding xe2x80x9cpreyxe2x80x9d proteins recited in Table 7, columns 2 and 6, respectively (the yeast orthologs of these proteins are recited in columns 1 and 5, respectively). In still other embodiments, the chimeric polypeptide contains regions of a first protein from yeast, and a second human ortholog protein, where the yeast proteins are selected from the xe2x80x9cbaitxe2x80x9d and corresponding xe2x80x9cpreyxe2x80x9d proteins recited in Table 7, columns 1 and 5, respectively, while the human ortholog proteins are selected from the xe2x80x9cbaitxe2x80x9d and corresponding xe2x80x9cpreyxe2x80x9d proteins recited in Table 7, columns 2 and 6, respectively.
In some embodiments, the chimeric polypeptide(s) of the complex include(s) six or more amino acids of a first protein covalently linked to six or more amino acids of a second protein. In other embodiments, the chimeric polypeptide includes at least one binding domain of a first or second protein.
Preferably, the chimeric polypeptide includes a region of amino acids of the first polypeptide able to bind to a second polypeptide. Alternatively, or in addition, the chimeric polypeptide includes a region of amino acids of the second polypeptide able to bind to the first polypeptide.
Nucleic acid encoding the chimeric polypeptide, as well as vectors and cells containing these nucleic acids, are within the scope of the present invention. The chimeric polypeptides can be constructed by expressing nucleic acids encoding chimeric polypeptides using recombinant methods, described above, then recovering the chimeric polypeptides, or by chemically synthesizing the chimeric polypeptides. Host-vector systems that can be used to express chimeric polypeptides include, e.g.: (i) mammalian cell systems which are infected with vaccinia virus, adenovirus; (ii) insect cell systems infected with baculovirus; (iii) yeast containing yeast vectors or (iv) bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.
The expression of the specific proteins may be controlled by any promoter/enhancer known in the art including, e.g.: (i) the SV40 early promoter (see e.g., Bernoist and Chambon, Nature 290: 304-310 (1981)); (ii) the promoter contained within the 3xe2x80x2-terminus long terminal repeat of Rous Sarcoma Virus (see e.g., Yamamoto, et al., Cell 22: 787-797 (1980)); (iii) the Herpesvirus thymidine kinase promoter (see e.g., Wagner, et al., Proc. Natl. Acad. Sci. USA 78: 1441-1445 (1981)); (iv) the regulatory sequences of the metallothionein gene (see e.g., Brinster, et al., Nature 296: 39-42 (1982)); (v) prokaryotic expression vectors such as the xcex2-lactamase promoter (see e.g., Villa-Kamaroff, et al., Proc. Natl. Acad. Sci. USA 75: 3727-3731 (1978)); (vi) the tac promoter (see e.g., DeBoer, et al., Proc. Natl. Acad. Sci. USA 80: 21-25 (1983)).
Plant promoter/enhancer sequences within plant expression vectors may also be utilized including, e.g.,: (i) the nopaline synthetase promoter (see e.g., Herrar-Estrella, et al., Nature 303: 209-213 (1984)); (ii) the cauliflower mosaic virus 35S RNA promoter (see e.g., Garder, et al., Nuc. Acids Res. 9: 2871 (1981)) and (iii) the promoter of the photosynthetic enzyme ribulose bisphosphate carboxylase (see e.g., Herrera-Estrella, et al., Nature 310: 115-120 (1984)).
Promoter/enhancer elements from yeast and other fungi (e.g., the Gal4 promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinase promoter, the alkaline phosphatase promoter), as well as the following animal transcriptional control regions, which possess tissue specificity and have been used in transgenic animals, may be utilized in the production of proteins of the present invention.
Other animal transcriptional control sequences derived from animals include, e.g.,: (i) the insulin gene control region active within pancreatic xcex2-cells (see e.g., Hanahan, et al., Nature 315: 115-122 (1985)); (ii) the immunoglobulin gene control region active within lymphoid cells (see e.g., Grosschedl, et al., Cell 38: 647-658 (1984)); (iii) the albumin gene control region active within liver (see e.g., Pinckert, et al., Genes and Devel. 1: 268-276 (1987)); (iv) the myelin basic protein gene control region active within brain oligodendrocyte cells (see e.g., Readhead, et al., Cell 48: 703-712 (1987)); and (v) the gonadotrophin-releasing hormone gene control region active within the hypothalamus (see e.g., Mason, et al., Science 234: 1372-1378 (1986)).
The vector may include a promoter operably-linked to nucleic acid sequences which encode a chimeric polypeptide, one or more origins of replication, and optionally, one or more selectable markers (e.g., an antibiotic resistance gene). A host cell strain may be selected which modulates the expression of chimeric sequences, or modifies/processes the expressed proteins in a desired manner. Moreover, different host cells possess characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, phosphorylation, and the like) of expressed proteins. Appropriate cell lines or host systems may thus be chosen to ensure the desired modification and processing of the foreign protein is achieved. For example, protein expression within a bacterial system can be used to produce an unglycosylated core protein; whereas expression within mammalian cells ensures xe2x80x9cnativexe2x80x9d glycosylation of a heterologous protein.
The invention further provides antibodies and antibody fragments (such as Fab or (Fab)2 fragments) that bind specifically to the complexes described herein. By xe2x80x9cspecifically bindsxe2x80x9d is meant an antibody that recognizes and binds to a particular polypeptide complex of the invention, but which does not substantially recognize or bind to other molecules in a sample, or to any of the polypeptides of the complex when those polypeptides are not complexed.
For example, a purified complex, or a portion, variant, or fragment thereof, can be used as an immunogen to generate antibodies that specifically bind the complex using standard techniques for polyclonal and monoclonal antibody preparation.
A full-length polypeptide complex can be used, if desired. Alternatively, the invention provides antigenic fragments of polypeptide complexes for use as immunogens. In some embodiments, the antigenic complex fragment includes at least 6, 8, 10, 15, 20, or 30 or more amino acid residues of a polypeptide. In one embodiment, epitopes encompassed by the antigenic peptide include the binding domains of the polypeptides, or are located on the surface of the protein, e.g., hydrophilic regions.
If desired, peptides containing antigenic regions can be selected using hydropathy plots showing regions of hydrophilicity and hydrophobicity. These plots may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, Proc. Nat. Acad. Sci. USA 78:3824-3828 (1981); Kyte and Doolittle, J. Mol. Biol. 157:105-142 (1982).
The term xe2x80x9cantibodyxe2x80x9d as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen, such as a polypeptide complex. Such antibodies include, e.g.,polyclonal, monoclonal, chimeric, single chain, Fab and F(abxe2x80x2)2 fragments, and an Fab expression library. In specific embodiments, antibodies to human ortholog complexes.
Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies. For example, for the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by injection with the native protein, or a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation can contain, for example, recombinantly expressed polypeptide complex. Alternatively, the immunogenic polypeptides or complex may be chemically synthesized, as discussed above. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, e.g., Freund""s (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents. If desired, the antibody molecules directed against complex can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
The term xe2x80x9cmonoclonal antibodyxe2x80x9d or xe2x80x9cmonoclonal antibody compositionxe2x80x9d, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of a polypeptide complex. A monoclonal antibody composition thus typically displays a single binding affinity for a particular protein with which it immunoreacts. For preparation of monoclonal antibodies directed towards a particular complex, or polypeptide, any technique that provides for the production of antibody molecules by continuous cell line culture may be utilized. Such techniques include, e.g., the hybridoma technique (see Kohler and Milstein, Nature 256: 495-497 (1975)); the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., Immunol Today 4: 72 (1983)); and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., (1985) pp. 77-96). If desired, human monoclonal antibodies may be prepared by using human hybridomas (see Cote, et al., Proc. Natl. Acad. Sci. USA 80: 2026-2030 (1983)) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., In: Monoclonal Antibodies and Cancer Therapy, supra).
Methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., Science 246: 1275-1281 (1989)) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for the desired protein or derivatives, fragments, analogs or homologs thereof. Non-human antibodies can be xe2x80x9chumanizedxe2x80x9d by techniques well known in the art. See e.g., U.S. Pat. No. 5,225,539. Antibody fragments that contain the idiotypes to a polypeptide or polypeptide complex may be produced by techniques known in the art including, e.g.: (i) an F(abxe2x80x2)2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(abxe2x80x2)2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) Fv, fragments.
Chimeric and humanized monoclonal antibodies against the polypeptide complexes, or polypeptides, described herein are also within the scope of the invention, and can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT International Application No. PCT/US86/02269; European Patent Application No. 184,187; European Patent Application No. 171,496; European Patent Application No. 173,494; PCT International Publication No. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Application No. 125,023; Better et al., Science 240: 1041-1043 (1988); Liu et al., Proc. Nat. Acad. Sci. USA 84: 3439-3443 (1987); Liu et al., J. Immunol. 139: 3521-3526 (1987); Sun et al., Proc. Nat. Acad. Sci. USA 84: 214-218 (1987); Nishimura et al., Cancer Res. 47: 999-1005 (1987); Wood et al., Nature 314: 446-449 (1985); Shaw et al., J. Natl. Cancer Inst. 80: 1553-1559 (1988); Morrison, Science 229: 1202-1207 (1985); Oi et al., BioTechniques 4: 214 (1986); U.S. Pat. No. 5,225,539; Jones et al., Nature 321: 552-525 (1986); Verhoeyan et al., Science 239: 1534 (1988); and Beidler et al., J. Immunol. 141: 4053-4060 (1988).
Methods for the screening of antibodies that possess the desired specificity include, e.g., enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art. For example, selection of antibodies that are specific to a particular domain of a polypeptide complex is facilitated by generation of hybridomas that bind to the complex, or fragment thereof, possessing such a domain.
In certain embodiments of the invention, antibodies specific for the polypeptide complexes described herein may be used in various methods, such as detection of complex, and identification of agents which disrupt complexes. These methods are described in more detail, below. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, xcex2-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.
Polypeptide complex-specific, or polypeptide-specific antibodies, can also be used to isolate complexes using standard techniques, such as affinity chromatography or immunoprecipitation. Thus, the antibodies disclosed herein can facilitate the purification of specific polypeptide complexes from cells, as well as recombinantly produced complexes expressed in host cells.
In a specific embodiment, the invention provides kits containing a reagent, for example, an antibody described above, which can specifically detect a polypeptide complex, or a constituent polypeptide, described herein. Such kits can contain, for example, reaction vessels, reagents for detecting complex in sample, and reagents for development of detected complex, e.g. a secondary antibody coupled to a detectable marker. The label incorporated into the anti-complex, or anti-polypeptide antibody may include, e.g., a chemiluminescent, enzymatic, fluorescent, colorimetric or radioactive moiety. Kits of the present invention may be employed in diagnostic and/or clinical screening assays.
The invention further provides pharmaceutical compositions of purified complexes suitable for administration to a subject, most preferably, a human, in the treatment of disorders involving altered levels of such complexes. Such preparations include a therapeutically-effective amount of a complex, and a pharmaceutically acceptable carrier. As utilized herein, the term xe2x80x9cpharmaceutically acceptablexe2x80x9d means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals and, more particularly, in humans. The term xe2x80x9ccarrierxe2x80x9d refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered and includes, but is not limited to such sterile liquids as water and oils.
The therapeutic amount of a complex which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and may be determined by standard clinical techniques by those of average skill within the art. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the overall seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient""s circumstances. [[However, suitable dosage ranges for intravenous administration of the complexes of the present invention are generally about 20-500 micrograms (xcexcg) of active compound per kilogram (Kg) body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.
Various delivery systems are known and can be used to administer a pharmaceutical preparation of a complex of the invention including, e.g.: (i) encapsulation in liposomes, microparticles, microcapsules; (ii) recombinant cells capable of expressing the polypeptides of the complex; (iii) receptor-mediated endocytosis (see, e.g., Wu et al., J. Biol. Chem. 262: 4429-4432 (1987)); (iv) construction of a nucleic acid encoding the polypeptides of the complex as part of a retroviral or other vector, and the like.
Methods of administration include, e.g., intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The pharmaceutical preparations of the present invention may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically-active agents. Administration can be systemic or local. In addition, it may be advantageous to administer the pharmaceutical preparation into the central nervous system by any suitable route, including intraventricular and intrathecal injection. Intraventricular injection may be facilitated by an intraventricular catheter attached to a reservoir (e.g., an Ommaya reservoir). Pulmonary administration may also be employed by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. It may also be desirable to administer the pharmaceutical preparation locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, by injection, by means of a catheter, by means of a suppository, or by means of an implant. In a specific embodiment, administration may be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.
Alternatively, pharmaceutical preparations of the invention may be delivered in a vesicle, in particular a liposome, (see, e.g., Langer, Science 249:1527-1533 (1990)) or via a controlled release system including, e.g., a delivery pump (see, e.g., Saudek, et al., New Engl. J. Med. 321: 574 (1989) and a semi-permeable polymeric material (see, e.g., Howard, et al., J. Neurosurg. 71: 105 (1989)). Additionally, the controlled release system can be placed in proximity of the therapeutic target (e.g., the brain), thus requiring only a fraction of the systemic dose. See, e.g., Goodson, In: Medical Applications of Controlled Release, 1984 (CRC Press, Bocca Raton, Fla.).
The invention further provides methods of identifying an agent which modulate formation or stability a polypeptide complex described herein. By modulate is meant to increase or decrease the rate at which the complex is assembled or dissembled, or to increase or decrease the stability of an assembled complex. Thus, an agent can be tested for its ability to disrupt a complex, or to promote formation or stability of a complex.
In one embodiment, the invention provides a method of identifying an agent that promotes disruption of a complex. The method includes providing a polypeptide complex, contacting the complex with a test agent, and detecting the presence of a polypeptide displaced from the complex. The presence of displaced polypeptide indicates the disruption of the complex by the agent. In some embodiments, the complex is a human ortholog complex, as described above, which includes xe2x80x9cbaitxe2x80x9d and xe2x80x9cpreyxe2x80x9d proteins selected from those recited in Table 7. In other embodiments, the complex contains at least one microtubule or microtubule-associated protein, as described above, and is selected from the complexes recited in Table 4. In other embodiments, the complex contains at least one heme biosynthesis protein, as described above, and is the complex recited in Table 5. In yet another embodiment, the complex contains at least one cell wall or cell wall-synthesis protein, as described above, and is selected from the complexes recited in Table 6. Agents which disrupt complexes of the invention may present novel modulators of cell processes and pathways in which the complexes participate. For example, agents which disrupt complexes involving microtubule proteins may be selected as potential anti-fungal therapeutics.
Any compound or other molecule (or mixture or aggregate thereof) can be used as a test agent. In some embodiments, the agent can be a small peptide, or other small molecule produced by e.g., combinatorial synthetic methods known in the art. Disruption of the complex by the test agent, e.g. binding of the agent to the complex, can be determined using art recognized methods, e.g., detection of polypeptide using polypeptide-specific antibodies, as described above. Bound agents can alternatively be identified by comparing the relative electrophoretic mobility of complexes exposed to the test agent to the mobility of complexes that have not been exposed to the test agent.
Agents identified in the screening assays can be further tested for their ability to alter and/or modulate cellular functions, particularly those functions in which the complex has been implicated. These functions include, e.g., control of cell-cycle progression; regulation of transcription; control of intracellular signal transduction, etc., as described in detail above.
In another embodiment, the invention provides methods for inhibiting the interaction of a polypeptide with a ligand, by contacting a complex of the protein and the ligand with an agent that disrupts the complex, as described above. In certain embodiments, the polypeptides are microtubule or microtubule-associated proteins, heme biosynthesis proteins, or cell wall or cell wall-synthesis proteins. In certain embodiments, the ligand is an interacting polypeptide, and the polypeptide and ligands are selected from those recited in Tables 4-6. Inhibition of complex formation allows for modulation of cellular functions and pathways in which the targeted complexes participate.
In another embodiment, the invention provides a method for identifying a polypeptide complex in a subject. The method includes the steps of providing a biological sample from the subject, detecting, if present, the level of polypeptide complex. In some embodiments, the complex includes a first polypeptide (a xe2x80x9cbaitxe2x80x9d polypeptide) selected from the polypeptides recited in Table 7, column 2, and a second polypeptide (xe2x80x9cpreyxe2x80x9d polypeptide) selected from the polypeptides recited in Table 7, column 6. Any suitable biological sample potentially containing the complex may be employed, e.g. blood, urine, cerebral-spinal fluid, plasma, etc. Complexes may be detected by, e.g., using complex-specific antibodies as described above. The method provides for diagnostic screening, including in the clinical setting, using, e.g., the kits described above.
In still another embodiment, the present invention provides methods for detecting a polypeptide in a biological sample, by providing a biological sample containing the polypeptide, contacting the sample with a corresponding polypeptide to form a complex under suitable conditions, and detecting the presence of the complex. A complex will form if the sample does, indeed, contain the first polypeptide. In some embodiments, the polypeptide being detecting is a xe2x80x9cpreyxe2x80x9d protein selected from the polypeptides recited in Table 7, column 6, and is detected by complexing with the corresponding xe2x80x9cbaitxe2x80x9d protein recited in Table 7, column 2. Conversely, in other embodiments the polypeptide being detected is the xe2x80x9cbaitxe2x80x9d protein. Alternatively, a yeast xe2x80x9cbaitxe2x80x9d or xe2x80x9cpreyxe2x80x9d ortholog may be employed to form a chimeric complex with the polypeptide in the biological sample.
In still another embodiment, the invention provides methods for removing a first polypeptide from a biological sample by contacting the biological sample with the corresponding second peptide to form a complex under conditions suitable for such formation. The complex is then removed from the sample, effectively removing the first polypeptide. As with the methods of detecting polypeptide described above, the polypeptide being removed may be either a xe2x80x9cbaitxe2x80x9d or xe2x80x9cpreyxe2x80x9d protein, and the second corresponding polypeptide used to remove it may be either a yeast or human ortholog polypeptide.
Methods of determining altered expression of a polypeptide in a subject, e.g. for diagnostic purposes, are also provided by the invention. Altered expression of proteins involved in cell processes and pathways can lead to deleterious effects in the subject. Altered expression of a polypeptide in a given pathway leads to altered formation of complexes which include the polypeptide, hence providing a means for indirect detection of the polypeptide level. The method involves providing a biological sample from a subject, measuring the level of a polypeptide complex of the invention in the sample, and comparing the level to the level of complex in a reference sample having known polypeptide expression. A higher or lower complex level in the sample versus the reference indicates altered expression of either of the polypeptides that forms the complex. The detection of altered expression of a polypeptide can be use to diagnose a given disease state, and or used to identify a subject with a predisposition for a disease state. Any suitable reference sample may be employed, but preferably the test sample and the reference sample are derived from the same medium, e.g. both are urine, etc. The reference sample should be suitably representative of the level polypeptide expressed in a control population.
In a certain embodiment, the polypeptide complex contains a xe2x80x9cbaitxe2x80x9d polypeptide selected from the polypeptides recited in Table 7, column 2, and a xe2x80x9cpreyxe2x80x9d polypeptide selected from the polypeptides recited in Table 7, column 6.
The invention further provides methods for treating or preventing a disease or disorder involving altered levels of a polypeptide complex, or polypeptide, disclosed herein, by administering to a subject a therapeutically-effective amount of at least one molecule that modulates the function of the complex. As discussed above, altered levels of polypeptide complexes described herein may be implicated in disease states resulting from a deviation in normal function of the pathway in which a complex is implicated. For example, altered levels of the observed complex between YGR010Wp and YLR328Wp may be implicated in disruptions in arginine metabolism, leading to retinal atrophy, for example. In subjects with a deleteriously high level of complex, modulation may consist, for example, by administering an agent which disrupts the complex, or an agent which does not disrupt, but down-regulates, the functional activity of the complex. Alternatively, modulation in subjects with a deleteriously low level of complex may be achieved by pharmaceutical administration of complex, constituent polypeptide, or an agent which up-regulates the functional activity of complex. Pharmaceutical preparations suitable for administration of complex are described above.
In one embodiment, a disease or disorder involving altered levels of a polypeptide selected from the polypeptides recited in Table 7, column 2 or the corresponding polypeptides in column 4, is treated by administering a molecule that modulates the function of the polypeptide. In certain embodiments, the modulating molecule is the corresponding polypeptide, e.g. administering a xe2x80x9cpreyxe2x80x9d protein corresponding to a xe2x80x9cbaitxe2x80x9d protein modulates the latter by forming a complex with it.
The details of one or more embodiments of the invention are set forth in the description above. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods and materials are now described. For example, additional interactions can be identified using other two-hybrid systems (i.e. using a LexA binding domain fusion or HIS3 as a reporter gene), including variables such as different protein domains or genomic activation domain libraries. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.